Microcapillary-based flow-through immunosensor and displacement immunoassay using the same

ABSTRACT

A displacement-type flow immunoassay is performed using a microcapillary  sage. The inner wall of the microcapillary passage has immobilized thereon antibodies to the antigen of interest. Labeled antigen is immunologically bound to the immobilized antibodies. Sample antigen passing through the column displaces the labeled antigen. Downstream, the displaced labeled antigen is detected. The microcapillary format of the present invention enhances the sensitivity of the immunoassay over the sensitivity of displacement-type flow immunoassays performed in a column at similar flow rates.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related generally to immunosensors, and morespecifically to immunosensors for flow-through displacementimmunoassays.

2. Description of the Background Art

Immunoassays exist in a variety of formats that utilize the interactionof antibodies with antigens usually including direct binding,competitive and sandwich assay schemes. The continuous flow immunoassayis a unique displacement assay that measures the dissociation of afluorescently labeled antigen from an antibody bound on a solid supportwhen the antigen flows past the antibody (U.S. Pat. No. 5,183,740,issued Feb. 2, 1993 to Ligler et al., the entirety of which isincorporated herein by reference for all purposes.) The displacement ofthe labeled antigen is proportional to the quantity of antigen presentin the sample. Sensitivity of the assay is dependent on the dissociationconstant of the antibody and the probability of antigen-to-antibodyinteraction in the flow system.

In previous studies on flow immunoassays using columns of packed beadsor porous membranes as substrates for the antibody immobilization, thefollowing parameters have been determined to affect signal magnitude andassay sensitivity:

(1) The affinity of the antibody for the antigen must be as high orhigher than the affinity of the antibody for the labeled analog underthe conditions of operation of the displacement immunoassay.

(2) There is a minimum number of antibody-labeled antigen complexes thatmust be present in the assay in order to generate a signal. Past thisminimum level, increasing the number of antibodies by increasing theamount of substrate or antibody density increases the signal magnitudeand the number of assays that can be performed, but may decrease theantigen sensitivity, probably due to rebinding of labeled antigen toimmobilized antibody. The minimum detectable amount of displaced labeledantigen is also a function of detector sensitivity.

(3) For each antigen-antibody pair, there seems to be an optimum flowrate which is probably related to the dissociation constant of theantibody. Increasing the flow rate above this level increasesspontaneous dissociation of labeled antigen, decreases antigen-antibodyinteraction time, and, consequently decreases the displacementefficiency (ratio of the number of moles of antigen added vs. number ofmoles of labeled antigen displaced). Decreasing the flow rate too muchresults in poor discrimination of the signal from background due to peakbroadening. In general, flow rates of 0.1 to 2.0 ml/min are taught with0.2 to 1 ml/min being preferable (U.S. Pat. No. 5,183,740, infra). Ifnot for an unacceptably low signal to background ratio, low flow rateswould be desirable for the detection of low analyte concentrations insmall (e.g., one picoliter to ten microliters) samples.

In Wemhoff et al. (Wemhoff, G. A., S. Y. Rabbany, A. W. Kusterbeck, R.A. Ogert, R. Bredehorst, and F. S. Ligler J. Immunol. Methods, 156,223-230, 1992), the concept of displacement efficiency was introduced asmeans for comparing assay performance as various parameters weremodified. The displacement efficiency is at its maximum when theconcentration of antigen added is low relative to the dynamic range forthe column being used. The amount of displaced labeled antigen moleculesdoes not exceed the displacement efficiency times the concentration oflabeled antigen bound to the immobilized antibody, even when highconcentrations of antigen are added. For the packed bed columns, amaximum displacement efficiency of about 0.001 was typical under optimalflow conditions.

U.S. Pat. No. 5,183,740 to Ligler et al. teaches a variety of supportmedia, including capillary tubes. That patent, however, attaches nocriticality to the form of the support media or column. Moreover, thatpatent fails to teach a range of capillary inner diameters and lengthsand fails to suggest any relationship between capillary diameter/lengthand sensitivity.

Finally, obtaining consistent results from sample to sample requirescolumns that can be manufactured consistently, reliably, andreproducibly. Packing columns with beads is an imprecise process thatresults in variability among columns.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to improve thesensitivity of a displacement-type flow immunoassay.

It is a another object of the present invention to reduce the amount ofsample required for a displacement-type flow immunoassay.

It is a further requirement of the present invention to provide a flowimmunoassay support that can be prepared easily and reproducibly.

It is yet another object of the present invention to provide a flowimmunoassay support that can be used with integrated optics.

These and other objects are accomplished by a microcapillary-based flowimmunoassay. An antibody is immobilized on the interior of amicrocapillary tube. The available antigen-binding sites of the antibodyare then immunologically bound to a labeled analog of the antigen. Whena sample containing the antigen flows through the microcapillary tube,sample antigen displaces the labeled analog.

If the microcapillary functioned as a support for the displacement assayin the same manner as packed beads, the operational parameters for thepacked bead columns would indicate that the microcapillary would notproduce sufficient signal for measurement. Based on this displacementefficiency and the estimated number of labeled antigen molecules in thecapillary, a maximum of 1.4×10⁻¹⁸ moles of labeled antigen could bedisplaced at any one sample addition. (The amount of labeled antigenthat can be displaced from the capillary is calculated based on thefollowing assumptions: (1) As described for the antibody immobilizationchemistry by Bhatia et al., Anal. Biochem, 178, 408-413, 1989), up to0.66 nm/mm² antibody can be immobilized on borosilicate glass. Theantibody can bind a small antigen in a 1:1 ratio. (2) The surface areaof the capillary is 346 mm². (3) The antibody has a molecular weight ofapproximately 160,000, and (4) the displacement efficiency is 0.001.Thus, 346 mm² * (0.66) ng antibody/mm²) * 1 nmole antibody/160,000 ng) *(nmole labeled antigen/1 nmole antibody/mm²) * 0.001=1.4×10⁻¹⁸ moles oflabeled antigen.) Assuming no peak broadening, this amount of antigen ina 100 μl volume would produce a molarity of 1.4×10⁻¹⁴, which would notbe detected using a standard HPLC fluorimeter with a sensitivity to thelabel of approximately 10⁻¹¹ molar. Nevertheless, as shown in theDetailed Description of the Preferred Embodiments below, the presentinvention actually provides enhanced sensitivity over prior artdisplacement immunoassays.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Preferred Embodimentsand the accompanying drawings in which like numerals in differentfigures represent the same structures or elements, wherein:

FIG. 1A is a simplified schematic of a microcapillary immunosensoraccording to an embodiment of the present invention. FIG. 1B is anexpanded schematic view of a section of microcapillary from theimmunosensor shown in FIG. 1A. FIG. 1C is a simplified schematic of amicrocapillary immunosensor on a chip according to another embodiment ofthe present invention.

FIG. 2A shows peaks due to displacement of Cy5-TNB upon injecting 100 μLof variable concentrations of TNT solution on an anti-TNT antibody11B3-coated microcapillary immunosensor (0.55 mm i.d.; 20 cm long).Injections 1-9 correspond to a TNT concentration of 0.25, 0.5, 1, 5, 25,50, 125, 250, and 500 ng/mL, respectively. FIG. 2B shows peaks resultingdue to displacement of Cy5-TNB upon injecting variable concentrations ofTNT solution on an anti-TNT antibody-coated microcapillary immunosensor(0.55 mm i.d.; 20 cm long). Injections 1-9 correspond to a TNTconcentration of 0.15, 0.3, 0.15, 6.25, 62.5, 125, 250, 500, and 1000ng/mL, respectively.

FIG. 3 is a graph showing the mean integrated area±S. E. (triplicatesamples) obtained upon injecting 100 μL at variable concentrations ofTNT solutions to an anti-TNT 11B3 antibody-coated microcapillaryimmunosensor (0.8 mm i.d.; 20 cm long). The inset represents the lineardynamic range for TNT assay using the anti-TNT 11B3 antibody-coatedmicrocapillary immunosensor.

FIG. 4A, FIG. 4B, and FIG. 4C show, respectively, the integrated area,peak maxima, and full width at half maximum (FWHM) of the resultingpeaks obtained upon injecting 100 μL of 1 pg/mL TNT solution to aanti-TNT antibody-coated microcapillary immunosensor (0.55 mm i.d., 20cm) as a function of the flow rate (μL/min) of the flow buffer.

FIG. 5 shows the mean integrated area±S. E. (triplicate samples) of thepeaks obtained upon injecting 100 μL of 5 ng/mL TNT solution to ananti-TNT antibody-coated microcapillary immunosensor (0.55 mm i.d., 20cm) as function of the length of the microcapillary.

FIG. 6 shows the integrated area under the peaks obtained upon injectingvariable concentration of TNT solution through an anti-TNTantibody-coated microcapillary immunosensor (O) (0.55 mm i.d., 20 cm)and a HPLC column (). The solid line represents the actualconcentration of the TNT solution prepared via diluting a stock TNTsolution which was injected in either system.

FIG. 7A is a graph of fluorescence intensity vs. time resulting fromdisplacement of Cy5-TNB from the TNT capillary. Injections 1-5correspond to a TNT concentration of 0.5, 5, 25, 50, and 250 ng/ml,respectively.

FIG. 7B shows a standard curve (integrated fluorescence intensity vs.concentration) for TNT in flow buffer using the multi analyte capillaryflow immunosensor system.

FIG. 7C is graph of fluorescence intensity vs. time resulting fromdisplacement of Cy5-RDX from the RDX capillary. Injections 1-5correspond to a RDX concentration of 0.5, 5, 25, 50, and 250 ng/ml,respectively.

FIG. 7D shows a standard curve (integrated fluorescence intensity vs.concentration) for RDX in flow buffer using the multianalyte capillaryflow immunosensor system.

FIG. 8A shows the integrated fluorescence units of the area under thepeaks in the TNT (▪) and RDX (□) capillaries resulting from injecting 5,50, and 10,000 ng/ml of TNT into the multianalyte capillary flowimmunosensor system.

FIG. 8B shows the integrated fluorescence units of the area under thepeaks in the TNT (▪) and RDX (□) capillaries resulting from injecting 5,50, and 10,000 ng/ml of RDX into the multianalyte capillary flowimmunosensor system.

FIG. 8C shows the integrated fluorescence units of the area under thepeaks in the TNT (▪) and RDX (□) capillaries resulting from injectingmixtures of TNT and RDX at various concentrations into the multianalytecapillary flow immunosensor system; Injections 1, 2, and 3 correspond toa 100:100, 100:500, and 150:5000 ng/ml mixture of TNT:RDX, respectively.Assay conditions: flow rate=100 μl/min; injection volume=200 μl;capillary i.d.=0.55 mm; capillary length=20 cm.

FIG. 9 is a simplified schematic representing a continuous flowimmunoassay on a chip.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

U.S. Pat. No. 5,183,740 to Ligler et al. describes the generalcharacteristics of displacement immunoassays under flow conditions andis incorporated herein, in its entirety, by reference for all purposes.

A microcapillary tube useful according to the present invention has aninner diameter of no greater than about 1 mm. At diameters greater thanabout 1 mm, the unexpectedly large increase in sensitivity will notoccur. There is no lower limit on the size of the inner diameter of themicrocapillary, as long as the relevant sample will flow through it.

For each antigen-antibody pair, there seems to be an optimum flow ratethat is related to the binding kinetics of the antibody. Increasing theflow rate above this level can both cause increased spontaneousdissociation of labeled antigen and decreased antigen-antibodyinteraction time. Decreasing the flow rate too much results in poordiscrimination of the signal from background due to peak broadening.When comparing flow streams through microcapillaries of different sizes,however, it is more accurate to refer to linear flow velocity ratherthan flow rate.

For the detection of TNT using antibodies as described in theaccompanying examples, the best results were obtained at linear flowvelocities (flow rate/cross-sectional area of the microcapillary) ofabout 20 cm/min to about 105 cm/min. Flow velocities of less than 20cm/min may provide equal or better results, but will require a pumpcapable of slower flow velocities than those used in the accompanyingexamples Although the optimum linear flow velocities for otherantigen/antibody pairs may vary somewhat from those described for TNTabove, the optimum flow velocities for many antigen/antibody pairsshould be near those described above for TNT detection. In any event, aperson skilled in the art of immunoassays should be able to determinethe actual optimal flow velocity for any antigen/antibody pairdetermined empirically, without the exercise of inventive skill, basedupon the teachings provided in the instant application.

The microcapillary tubes may be made from any material that does notabsorb or change the chemical properties of the antigens, labels orantibodies used and is capable of immobilizing an antibody thereon.Typically, the microcapillary tubes are made of glass, but they may alsobe made, for example, from a polymer (such as polystyrene,polycarbonate, polyvinyl and polyacrylic) or ceramic.

As shown in FIG. 1A, pump 10 delivers flow buffer 12 to injector 14. Aliquid sample suspected of containing antigen is injected into flowbuffer 12 by injector 14. From injector 14, the flow buffer/samplestream passes through microcapillary 16. As shown in FIG. 1B, innerwalls 30 of capillary 16 have antibodies 32 immobilized (e.g., bycovalent bonding) thereon. The antigen-binding sites of antibodies 32are effectively saturated (i.e., the binding sites are at leastsufficiently close to saturation with labeled analog molecules that themicrocapillary essentially behaves as though the binding sites werecompletely saturated. Throughout the present specification and claims,the term "saturated" encompasses "effectively saturated" unlessotherwise stated) with labeled analog 34 of the antigen of interest.Within microcapillary 16, unlabeled antigen, if present, displaceslabeled analog 34 into the stream of flow buffer 12. Downstream ofmicrocapillary 16, detector 18 detects displaced labeled analog 34. Thesignal from detector 36 is then fed into integrator 20, which integratesthese signals. The integrated signal may then be recorded. Waste fluidcontaining the sample and displaced labeled analog 34 are then collectedor drained into waste disposal 22.

The present invention may also be adapted to simultaneously test severalsamples and/or to test one sample for a plurality of antigens. In theseembodiments, a single pump and flow buffer stream are connected inparallel to several microcapillary tubes, each microcapillary tubehaving its inside surface coated with an antibody to a specific antigenof interest, the antibodies coated on the inside surface of eachmicrocapillary tube having their antigen binding sites saturated with alabeled analog of the corresponding antigen.

The small diameter of the microcapillary tubes used in the presentinvention allows for another novel immunoassay format. In this newformat, microcapillary passages, as well as an entrance and exit forflow buffer, are molded or micro-machined into a monolithic substratesuch as a chip or cartridge. If desired, the microcapillary passages maybe coiled or serpentine. The chip/cartridge may be made from anymaterial, for example, silicon or other semiconductor, polymer, orglass, that is neutral to the materials being analyzed, and that can bereadily machined or molded to form microcapillary passages thereinhaving inner walls capable of immobilizing an antibody. If desired, anelectroosmotic pump may be used to flow fluid through the microcapillaryor microcapillaries in the chip/cartridge (see Effenhauser, C. S., A.Manz, and H. M. Widmer. Analytical Chemistry. 67, 2284-2287, 1995.;Jacobson, S. C. and J. M. Ramsey. Analytical Chemistry, 68, 720-723,1996). The entireties of each of these papers are incorporated herein byreference for all purposes). Alternatively, pneumatic pumps ormechanical and valves could be used for fluid control and still producea small, lightweight flow immunosensor.

After machining or molding, the inner passages of the chip/cartridge arethen coated with antibody molecules immobilized thereon. The antigenbinding sites of these immobilized antibodies are then saturated with alabeled analog of the antigen. The finished assembly may then beinserted into an receptacle designed so that the entrance port of thechip/cartridge aligns and forms a releasable fluid-tight seal along aflow path for buffer downstream of a sample injector and upstream from adetector. If a detection cell/cuvette is external (i.e., in thereceptacle) to the chip, the exit port of the chip/cartridge should alsoalign with and form a releasable fluid-tight seal with the flow pathupstream from the detection cell/cuvette, but downstream from theentrance port. Where the detection cell is integrated with the chip, theexit port may be in fluid connection with the microcapillary passage,downstream of the entrance port, and should drain spent buffer and labelaway from the receptacle, or at least away from components of thereceptacle that may not be readily cleaned or replaced. Buffer flowthrough the microcapillary passage(s) is then established. Thereafter,sample is injected into the buffer flow and enters the microcapillarypassage(s) in the chip/cartridge. After exiting the microcapillarypassage, buffer flow and displaced labeled analog flow downstream intothe detector. The detector may be integrated into the chip/cartridge, orpositioned downstream from the chip/cartridge. After the assay has beencompleted, the chip/cartridge may be readily removed and a newchip/cartridge inserted. This embodiment forms a particularly usefularrangement for field use.

FIG. 1C shows a flow immunosensor chip 100. Buffer flow throughmicrocapillary passage 102 by virtue of an electroosmotic gradientgenerated by electrodes 104 and oppositely charged electrodes 106.Sample port 108 connects to microcapillary passage 102, allowing aliquid sample to be introduced into microcapillary passage 102.Downstream of sample port 108, microcapillary passage 102 divides intothree daughter microcapillary passages 110, 112, and 114. Each of thesethree daughter passages 110, 112, and 114 has, immobilized on its innersurface, a coating of an antibody that has its antigen recognition sitessaturated with a labeled analog of an antigen of interest. Theantibody/antigen pairs and labels used may be the same for each daughterpassage, or may differ among the daughter passages. Further downstream,at point 116, the inner coatings along the daughter passages terminate.Downstream of point 116, daughter passages 110, 112, and 114 rejoin toform passage 118 leading into detection cell/cuvette 120 (A chip cuvetteis disclosed in, for example, Liang, Z., N. Chiem, G. Ocvirk, T. Tang,K. Fluri, and K. J. Harrison. Analytical Chemistry, 68, 1040-1046, 1996,the entirety of which is incorporated herein by reference for allpurposes). The detection includes at least one window transparent toexcitation light and at least one window transparent to the fluorescentemissions of the excited label.

Optical fiber 122 transports excitation light from light source 124 (forexample, an LED) to optical fiber 128 through releasable coupler 126. Asimple butt coupling (not shown) may be used rather that couple 126.Optical fiber 128 transports the excitation light to detection cell 120.The excitation light causes any displaced labeled analog in detectioncell 120 to emit fluorescent light. Optical fiber 130 transports thisfluorescent emission to detector 134 through releasable coupler 132 andoptical fiber 136. Appropriate filters for the excitation light andfluorescent emissions may be added at any point along the respectivelight paths. For example, filters may be incorporated into the ends ofdetection cell 120, between light source 124 and optical fiber 122,and/or between detector 134 and optical fiber 136. Releasable couplers126 and 132 are designed to optically couple fibers pairs 122/128, and130/136, respectively, when chip 100 is inserted into its receptacle(not shown). Power for an electroosmotic pump or other fluid pump, aswell as any other on-chip components, may be provided by a batteryincorporated into the chip or external to the chip.

In the embodiment shown in FIG. 1C, the light source and detector, andassociated circuitry for those components, are included in a receptacleexternal to the chip. That arrangement decreases the cost of the chip,making the disposability of the chip highly practical. If desired,however, an LED, and any appropriate filters, may be incorporated intothe chip as the light source, and a photodiode, and any appropriatefilters, may be provided as the detector. Data from a detector internalto the chip could be ported to a recording device and/or computer via,for example, an RS232 port built onto the chip. The circuitry for eachof these components may be provided on the chip. The cost of theseadditional on-chip components and their required circuitry, of course,significantly adds to the expense of the chip.

Having described the invention, the following examples are given toillustrate specific applications of the invention including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

EXAMPLES Example 1 Displacement immunoassay in a microcapillary

The following materials, equipment, and methods were used in allexamples, unless otherwise stated:

Materials. Fused silica microcapillaries (Polymicro Technologies, Inc.);3-mercaptopropyltrimethoxysilane (MTS),N-succinimidyl-4-maleimidobutyrate (GMBS; Fluka); ethanol (200 proof;Warner-Graham); toluene (Mallinkrodt); tween, (Sigma); sodiummonophosphate (Aldrich); 2,4,6-trinitrotoluene (TNT), and1,3,5-trinitrobenzene (TNB). Anti-TNT antibody 11B3 was generated at NRLand another anti-TNT antibody was purchased from Strategic Diagnostics,Inc. All reagents were used as received without further purification andaqueous solutions were prepared in doubly distilled deionized waterunless otherwise noted.

Equipment. FIG. 1A schematically illustrates the microcapillary-basedcontinuous flow immunosensor used in these examples. A Rabbit-Plusperistaltic pump (Rainin Instruments), a low pressure Rheodyne five-wayvalve sample injector, a Model 821-FP spectrofluorometer (Jasco, Inc.),and a HP33936B Series II integrator (Hewlett-Packard) were used. A modelHP-9114B disc drive (Hewlett-Packard, Inc.) was utilized to store analogoutput data from the fluorometer for subsequent analysis. All connectingtubes were 0.3-mm-i.d. Teflon (Cole Palmer).

Flow Buffer. The flow buffer comprised of a mixture of sodiummonophosphate (10 mM), ethanol (2.5%), and Tween-20 (0.01%, Aldrich).All solutions to be analyzed using the microcapillary immunosensor wereprepared in the flow buffer.

Antibody immobilization in a microcapillary. Antibodies were immobilizedon the inner walls of the microcapillaries essentially according to themethod of Bhatia, S. K., L. C. Shriver-Lake, K. J. Prior, et al. (Anal.Biochem, 178, 408-413, 1989). A desired length of fused silicamicrocapillary (0.80 or 0.55 mm i.d.) was incubated with a 4% MTSsolution (in toluene) at room temperature for one hr. After flushing themicrocapillary with toluene 3 times, a 2 mM crosslinker solution (GMBS)was introduced in the microcapillary and incubated at room temperaturefor 1 hr. The microcapillary was then rinsed with deionized water. A 1mg/ml solution of anti-TNT antibody 11B3 or Strategic Diagnosticsanti-TNT antibody was introduced into the microcapillary for 1 hr. Themicrocapillary was then rinsed with water 3 times. Finally, a 30 μMCy5-trinitrobenzene (Cy5-TNB; synthesized as previously described (Bart,J. C., L. L Judd, K. E. Hoffman, A. M. Wilkins, P. T. Charles and A. W.Kusterbeck, ACS Symposium Series, in press) solution was introduced intothe microcapillary and incubated in a refrigerator at 4° C. for about 8to 12 hours. (No attempt was made to determine the minimum usefulincubation time. Far shorter incubation times than those used in thepresent examples may be empirically determined to provide equivalentresults)

Immunoassay. The flow buffer was pumped through the microcapillary atthe desired flow rate and the fluorescence of the Cy5-TNB was monitoreddownstream using the fluorometer equipped with a 16 μL flow cell. Unlessotherwise stated, the microcapillary was 20 cm long with a 0.55 mm i.d.and was run at a flow rate of 0.2 ml/min (linear flow velocity=84cm/min). The excitation wavelength used was 632±4 nm and thefluorescence emission was monitored at 662±4 nm. Initially, there was aconstantly changing slope for background fluorescence intensity as afunction of time indicating the washing of the excess andnonspecifically adsorbed Cy5-TNB from the walls of the microcapillary.After approximately 30 min, the background fluorescence stabilized, i.e.a constant rate for change in fluorescence intensity was achieved. Atthis point, 100 μL injections of flow buffer containing 0-500 ng/mL TNTwere made. The resulting peaks due to the displacement of thefluorescently-labeled antigen were recorded and the area under the peakswas quantified. Injections at each concentration were made intriplicate.

FIG. 2A and FIG. 2B represent the resulting peaks upon injecting TNT atvarious concentrations (0.015-1000 ng/mL) to an anti-TNT antibody 11B3coated microcapillary (FIG. 2A) and a Strategic Diagnosticanti-TNT-coated microcapillary (FIG. 2B). Injections 1-9 in FIG. 2Acorrespond to a TNT concentration of 0.25, 0.5, 1, 5, 25, 50, 125, 250,and 500 ng/mL, respectively. Injections 1-9 in FIG. 2B correspond to aTNT concentration of 0.15, 0.3, 0.15, 6.25, 62.5, 125, 250, 500, and1000 ng/mL, respectively. FIG. 3 shows the mean integrated area±S. E. oftriplicate assays resulting from displacement of Cy-TNB from the 11B3coated microcapillary immunosensor upon injecting varying concentrationsof TNT. These results clearly demonstrate that: (1) microcapillarybiosensor has a limit of detection which is at least three orders ofmagnitude better than packed bead (U.S. Pat. No. 5,183,740) and membranebased continuous flow immunosensors ("Displacement Assay on a PorousMembrane" F. S. Ligler, A. W. Kusterbeck, and S. Y. Rabbany, N. C.77,298, U.S. Application); (2) there is a much higher displacement oflabeled antigen observed for Strategic Diagnostic anti-TNT coatedmicrocapillary immunosensor compared 11B3 coated microcapillary which isconsistent with our ELISA results; and (3) under the currentexperimental conditions, the linear dynamic range is between 1-250 ng/mL(FIG. 3 inset).

Example 2 Effect of microcapillary diameter.

The effect of microcapillary diameter was studied using a 14 cm (0.80 mmi.d.) and a 20 cm (0.55 mm i.d.) microcapillaries which were coated with11B3 anti-TNT antibody using the immobilization protocol explainedabove. The length of the two microcapillaries was kept such that thetotal surface area available for antibody immobilization is constant(346 mm²). The limit of detection for TNT using both themicrocapillaries was then determined. The linear flow velocity for thetwo microcapillaries was kept constant. It was observed that the limitof detection for the 0.8 mm i.d. microcapillary was 15 pg/mL and thatfor 0.55 mm i.d. microcapillary was 1 pg/mL. This experiment clearlydemonstrates that the sensitivity of the system is dependent on thediameter of the microcapillary used. In this example, microcapillarieswith i.d. lower than 0.55 mm could not be used because a continuous flowat lower flow rates could not be achieved using our peristaltic pump.This is a limit of the current experimental set up but in principle,smaller i.d. microcapillaries should work better.

Example 3 Effect of flow rate

The effect of the flow rate was investigated using the 0.55 mm i.d.microcapillary (20 cm long). The flow rates ranged from 0.05 to 0.55ml/min (linear flow velocity range=21 cm/minute to 231 cm/minute). FIG.4A, FIG. 4B, and FIG. 4C represent the performance of the StrategicDiagnostic anti-TNT-coated microcapillary immunosensors as a function ofthe flow rate upon injecting 100 μL of 1 pg/mL TNT solution. FIG. 4Ademonstrates that the signal magnitude produced by the microcapillary ishighly dependent on the flow rate. Not only the fluorescence intensitybut also the peak maxima (FIG. 4B) and full width at half maximum of theresulting peaks (FIG. 4C) are also affected by the flow rate. Theconcentration tested, 1 pg/ml, can be detected at flow rates less than100 μL/min (linear flow velocity=42 cm/min), but is below the limit ofdetection for faster flow rates.

Example 4 Effect of microcapillary length

The effect of length of the microcapillary was investigated by comparingthe response of Strategic Diagnostic anti-TNT antibody coated 0.55 mmi.d. microcapillary as function of the length of the microcapillary. Thelinear flow velocity in all cases was kept constant at 190 μL/min(linear flow velocity=80 cm/min). FIG. 5 represents the integrated areaof the peaks upon injecting 100 μL plugs of 5 ng/mL TNT solution to themicrocapillary immunosensor. The magnitude of the displacement signal isdependent on the length of the microcapillary.

Example 5 Antigen quantitation

In order to investigate the viability of this system for thequantitation of antigen, we compared the results of TNT spiked samplesobtained by our microcapillary immunosensor and EPA method 8330 (HPLCmethod). For this experiment, a 1000 ng/mL stock TNT solution wasprepared in the flow buffer. One portion of the stock TNT solution wasdiluted using the flow buffer for microcapillary immunosensorquantitation and the other portion was diluted using the mobile phaseused for the HPLC. FIG. 6 is a plot of the results obtained using themicrocapillary immunosensor () and HPLC method (∘) as function of theactual concentration of TNT prepared using the stock TNT solution. Theline is a reference line indicating a 100% correlation between theexperimental results and the actual TNT concentration.

There are a few issues not apparent in FIG. 6 which require additionaldiscussion. First, the limit of detection for the EPA 8330 HPLC methodis 5 ng/mL whereas for the microcapillary immunosensor, it is 1 pg/mL.Second, up to 300 ng/mL, the correlation between the actual andexperimentally determined TNT concentration is better for themicrocapillary immunosensor compared to EPA 8330 HPLC method. Third,there is a negative bias for the microcapillary immunosensor forconcentrations above 300 ng/mL which is attributed to a reduction indisplacement immediately after a large displacement. It should be notedthat one can theoretically correct for the reduced displacement asdescribed in Rabbany et al. (Rabbany, S. Y., A. W. Kusterbeck, R.Bredehorst, and F. S. Ligler, Sensors & Actuators B, 29, 72-78, 1995.)or Yu et al. (Yu, K., A. W. Kusterbeck, J. P. Whelan, M. Hale and F. S.Ligler, Biosensors and Bioelectronics, 11, 725-734, 1996.).

Example 6 Displacement efficiency

This example investigated the displacement efficiency (D_(e)) of themicrocapillary- and packed column-based continuous flow immunosensors.D_(e) is defined as:

D_(e) =moles of Ag* detected/moles of Ag injected

where Ag* and Ag represent the Cy5-TNB and TNT, respectively. Usingmicrocapillaries 0.80 mm i.d. and 20 cm long, the displacementefficiencies of the Strategic Diagnostic and 11B3 anti-TNTantibody-coated microcapillaries were measured to be 0.8 and 0.4,respectively. In contrast, the displacement efficiency of a packedcolumn-based displacement continuous flow immunosensor using the 11B3anti-TNT antibody was measured at 0.0011. It should be noted that thecalculated amount of antibody immobilized on the microcapillary andpacked column were 0.002 nmoles¹ and 3.1 nmoles², respectively. Threepoints are clear from this study; (1) Strategic Diagnostic anti-TNTantibody microcapillary is more efficient by a factor of 2 compared tothe 11B3 anti-TNT antibody coated microcapillary immunosensor, (2) themicrocapillary immunosensor is more than 2 orders of magnitude moreefficient than the packed column immunosensor, and (3) themicrocapillary immunosensor requires much less antibody to generate asignal.

Example 7 Simultaneous detection of two different analytes

This example demonstrated that the capillary immunosensor format issuitable for use in the development of a multianalyte detection system.Toward this end, two capillaries were connected in parallel using a "T"connector and the simultaneous detection of two explosives, TNT and RDX,was demonstrated. Specifically, two capillaries, one coated withantibodies specific for 2,4,6-trinitrotoluene (TNT) and the otherspecific for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were combinedinto a single device to develop a multianalyte capillary flowimmunosensor. The fused silica capillaries were prepared by coatinganti-TNT and anti-RDX antibodies onto the silanized inner walls using aheterobifunctional crosslinker. After immobilization, the antibodieswere saturated with a suitable fluorophore-labeled antigen. A "T"connector was used to continuously flow the buffer solution through theindividual capillaries. To perform the assay, an aliquot of TNT, RDX, ora mixture of the two analytes was injected into the continuous flowstream. In each capillary, the target analyte displaced thefluorophore-labeled antigen from the binding pocket of the antibody. Thelabeled antigen displaced from either capillary was detected downstreamusing two portable spectrofluorimeters. Such a multianalyte approachalso offers an on-line test for cross-reactivity.

FIG. 7A through FIG. 7D represents the resulting peaks (FIG. 7A and FIG.7C) and the integrated fluorescence intensity (FIG. 7B and FIG. 7D) uponinjecting a mixture of RDX and TNT prepared in flow buffer at variousconcentrations (0.5-500 ng/ml) into the multianalyte capillary flowimmunosensor system. FIG. 7A (TNT capillary) and 7C (RDX capillary)represent the resulting peaks upon injecting a mixture of TNT and RDX tothe MCFI system. We also injected mixtures of TNT and RDX at lowerconcentrations (<0.5 ng/ml) and observed that the limit of detection forTNT and RDX in the multianalyte format at 100 μl/min flow rate were 0.1and 0.5 ng/ml, respectively. In both cases, there was a loss insensitivity by a factor of 5 compared to the individual capillaryimmunosensors described in an earlier section. This loss in sensitivitywas attributed to the dilution and peak broadening effect uponincorporating a "T" connector and other tubing connections in order todirect the flow into the two individual capillaries. This loss is alimitation of the experimental set up and with better connection tubingof low dead volume, the sensitivity can be improved. FIG. 7B and FIG. 7Drepresent the dynamic range of the TNT and RDX in the multianalyteformat. As observed in the individual capillary immunoassay, there was alinear dependency on the concentration of TNT (r² =0.95) and RDX (r²=0.96) up to 100 and 300 ng/ml, respectively, after which the signalsaturated in both cases.

A very important feature of a multianalyte sensor is the crossreactivityof multiple antigens with the immobilized antibodies. In order toinvestigate this issue, first injected TNT was first injected only in tothe multianalyte capillary flow immunosensor system and observed lessthan 1% displacement from the RDX capillary compared to the signal fromthe TNT capillary (FIG. 8A). Upon injecting RDX only to the multianalytecapillary flow immunosensor system, less than 1% signal was observed inthe TNT capillary compared to the signal from the RDX capillary FIG.8B). Finally, mixtures of TNT and RDX were injected and displacementfrom both the capillaries was observed (FIG. 8C). These resultsdemonstrate that there is minimal crossreactivity for the two antigensin the multianalyte capillary flow immunosensor system, so that it is aneffective means of analyzing samples containing mixtures of TNT and RDX.

Example 8 Capillary flow immunoassay in very small i.d. capillaries

As mentioned in the example 2, it was not possible to use capillariessmaller than 0.55 mm i.d. because of the peristaltic pump used. In orderto achieve a continuous flow in the smaller i.d. capillaries (0.05 mm),a syringe pump was used. For very efficient excitation and fluorescencecollection, the experimental set up under sheath flow conditions asdescribed by Cheng et al., Anal. Chem. 1990, 62, 496-503, was used.Briefly, fluorescence was measured downstream from the column using a635 nm laser (2.5 mW) for excitation and detecting the fluorescencesignal using a 682±11 nm band pass and a photomultiplier tube (PMT).

Anti-TNT antibody/Cy5-TNB complex was immobilized onto the inner wallsof 0.05 mm i.d. capillaries using the same protocol as used for thelarger diameter capillaries described in an earlier section. Flow bufferwas continuously flowed through the capillary using a syringe pump at aflow rate of 680 nl/min. Injections of 1.5 nl of TNT (concentrationranging from 1-10,000 ng/ml) were made into the capillary. Based onpreliminary experiments, the limit of detection for TNT under theabove-mentioned conditions was 50 ng/ml. Various parameters such as flowrate, antibody density, and injection volume were not optimized. But, itwas demonstrated that small i.d. capillaries (0.05 mm i.d.) can be usedfor the continuous flow immunoassay. Such small i.d. capillaries arevery flexible and can be packaged into a small portable unit.

Example 9 Continuous flow immunoassay on a chip

Preliminary experiments demonstrate the feasibility of the continuousflow immunoassay in a chip format. The experimental set up is shown inFIG. 9. Glass slides 200, 201 were etched to form a 5 cm long, 250 μmwide, and 80 μm deep channel (not shown) on each. This was followed byfusing slides 200 and 201 such that the on either slide formed a closedpath 202. Anti-TNT antibody/Cy5-TNB complex (not shown) was immobilizedonto the walls of path 202 using the same protocol as used for thecapillaries. Online detection at the end of etched path was achievedusing the set up described by Jed Harrison et al., Science, 1993, 261,895-896; Harrison et al., Sensors and Actuators B, 1996, 33, 105-109. A630 nm laser beam 204 (2.5 mW output) was made incident on the etchedpath at a 45° angle from the plane of the glass chip. A 682±11 nm bandpass filter and a PMT (not shown) placed at 90° from the plane of thechip were used to collect the fluorescence signal 206.

To perform an assay, flow buffer was continuously flowed through path202 at a flow rate of approximately 20 μl/min. 1 μl injections of TNTsolutions (concentration ranging from 50 pg/ml-1000 ng/ml) were madeinto the etched path of the chip using a 10 gl syringe 208. A lowerlimit of detection of 220 attomoles of TNT (1 μl of 50 pg/ml TNTsolution) was observed under these conditions. This system by no meanswas optimized but the proof of principle that such an immunoassay can beperformed on a chip format is established.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

What is claimed is:
 1. A flow-through immunosensor for performing adisplacement assay, comprising:one or more microcapillary passageshaving an inner diameter of less than about 1 mm, each of said one ormore microcapillary passages having an antibody immobilized on its innerwall, and a labeled analog of an antigen immunologically bound to, andsaturating antigen-binding sites of, said immobilized antibody, thusforming an antigen/labeled antibody pair bound to said inner wall ofsaid microcapillary passage; a sample introduction port at which aliquid sample suspected of containing said antigen may be introducedinto said one or more microcapillary passages; a detector downstreamfrom said sample introduction port and joined to said one or moremicrocapillary passages, said detector detecting said labeled analogdisplaced from said antibody; wherein, if said imnmunosensor comprisesmore than one microcapillary passages, each of said microcapillarypassages has bound to its inner wall an antigen/labeled antibody pairdifferent from that bound to that of any other of said microcapillarypassages.
 2. The flow-through immunosensor of claim 1, furthercomprising a flow device which causes said sample to flow from saidsample introduction port to said detector.
 3. The flow-throughimmunosensor of claim 1, wherein said flow device causes said sample toflow from through said microcapillary passage at a linear flow velocityof about no more than about 105 cm/min.
 4. The flow-through immunosensorof claim 1, wherein said microcapillary passage is a tube made of glassor polymer.
 5. The flow-through immunosensor of claim 4, wherein saidmicrocapillary passage is made of glass.
 6. The flow-throughimmunosensor of claim 1, wherein said antibody is specific for anexplosive.
 7. The flow-through immunosensor of claim 1 comprising nomore than one said microcapillary passage.
 8. The flow-throughinmunosensor of claim 1, wherein said flow device causes said sample toflow from through said microcapillary passage at a flow rate of 100μl/min or less.
 9. The flow-through immunosensor of claim 8, whereinsaid flow device causes said sample to flow from through saidmicrocapillary passage at a flow rate of 50 μl/min or less.
 10. A methodof performing a displacement assay using a flow-through immunosensor,comprising the steps of:establishing a buffer flow through one or moremicrocapillary passages, each of said microcapillary passages having aninner diameter of less than about 1 mm, an antibody immobilized on itsinner wall, and a labeled analog of an antigen immunologically bound to,and saturating antigen-binding sites of, said immobilized antibody, thusforming an antigen/labeled antibody pair bound to said inner wall ofsaid microcapillary passage; introducing a liquid sample, said samplebeing suspected of containing said antigen, into an introduction port insaid microcapillary passage; detecting, in a detector downstream fromsaid introduction port and joined to said microcapillary passage, saidbound labeled analogs displaced from said microcapillary passage by anymolecules of said antigen present in said sample; wherein, if saidimmnunosensor comprises more than one microcapillary passages, each ofsaid microcapillary passages has bound to its inner wall anantigen/labeled antibody pair different from that bound to that of anyother of said microcapillary passages.
 11. The method of claim 10,wherein said buffer flow has a linear flow velocity of about no morethan about 105 cm/min.
 12. The method of claim 10, wherein saidmicrocapillary passage is a tube made of glass or polymer.
 13. Themethod of claim 12, wherein said microcapillary passage is made ofglass.
 14. The method of claim 10, wherein said antigen is an explosive.15. The method of claim 10, wherein said buffer flow has a flow rate of100 μl/min or less.
 16. The method of claim 10, wherein said buffer flowhas a flow rate of 50 μl/min or less.